The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are considered “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA; 18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3). Additionally PUFA's are constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols. PUFA's are necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair and, are precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Studies have shown that a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin. Nutr., 28:958-966 (1975); Dyerberg, J. et al., Lancet, 2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., World Rev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet, 88:90-99 (2001)). The literature reports wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
A variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production. Genetic engineering has demonstrated that the natural abilities of some hosts can be substantially altered to produce various long-chain ω-3/ω-6 PUFAs. For example, production of arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) all require expression of either the Δ9 elongase/Δ8 desaturase pathway or the Δ6 desaturase/Δ6 elongase pathway. The Δ9 elongase/Δ8 desaturase pathway is present for example in euglenoid species and is characterized by the production of eicosadienoic acid [“EDA”; 20:2 ω-6] and/or eicosatrienoic acid [“ETrA”; 20:3 ω-3]. (FIG. 1). The Δ6 desaturase/Δ6 elongase pathway is predominantly found in algae, mosses, fungi, nematodes and humans and is characterized by the production of γ-linoleic acid [“GLA”; 18:3 ω-6] and/or stearidonic acid [“STA”; 18:4 ω-3]) (FIG. 1).
For some applications the Δ9 elongase/Δ8 desaturase pathway is favored. However Δ8 desaturase enzymes are not well known in the art leaving the construction of a recombinant Δ9 elongase/Δ8 desaturase pathway with limited options. The few Δ8 desaturase enzymes identified thus far have the ability to convert both EDA to dihomo-γ-linolenic acid [20:3, DGLA] and ETrA to eicosatetraenoic acid [20:4, ETA] (wherein ARA are EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a Δ5 desaturase, while DHA synthesis requires subsequent expression of an additional C20/22 elongase and a Δ4 desaturase).
Several Δ8 desaturase enzymes are known and have been partially characterized (see for example Δ8 desaturases from Euglena gracilis Wallis et al., Arch. Biochem. and Biophys., 365(2):307-316 (May 1999); WO 2000/34439; U.S. Pat. No. 6,825,017; WO 2004/057001; WO 2006/012325; WO 2006/012326). Additionally WO 2005/103253 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a Δ8 desaturase enzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885). Sayanova et al. (FEBS Lett., 580:1946-1952 (2006)) describes the isolation and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C20 Δ8 desaturase. Furthermore, commonly owned and co-pending U.S. Provisional Application No. 60/795,810 filed Apr. 28, 2006 discloses amino acid and nucleic acid sequences for a Δ8 desaturase enzyme from Pavlova lutheri (CCMP459).
A need remains therefore for additional Δ8 desaturase enzymes to be used in recombinant pathways for the production of PUFA's. Applicants have solved the stated need by developing a synthetically engineered mutant Euglena gracilis Δ8 desaturase.